Progress in Gene Therapies

In the November issue, we examined the acceleration in genome sequencing and the development of tests for genetic markers in people. Our focus this month is on achievements in therapies to cure or overcome genetic abnormalities and the diseases to which they give rise, as reported in the past year.

Though not exhaustive, the list is impressive, and despite the “more research required” refrain attached to many of the advances, there is a discernible and quickening trend of gene therapy moving “from bench to bedside.” Several very important implications for health policy, administration, education, and practice ensue. We will write about those implications separately; here, we simply rejoice in the beneficent implications for patients suffering from various conditions, from blindness to aging.

AgingMaintaining a really strict diet is known to extend lifespan and reduce the incidence of age-related diseases in organisms ranging from yeast to you and me. Jocelyn Rice, writing in Technology Review, reports that researchers recently noticed that young mice with a disabled version of a protein called S6K1 were leaner and had greater insulin sensitivity than normal mice. So, they bred two large groups of “knockout” mice that lacked a functional version of the gene for S6K1. Here’s how Rice described it:

“One group lived out their lives undisturbed, providing a measure of the group’s natural lifespan. The other group was put through extensive testing of cognitive and motor performance and metabolic health.

In female mice, the results were profound. Knockout females lived substantially longer than their normal counterparts. At 600 days–the mouse equivalent of human middle age–they excelled at motor performance tests, outdoing normal mice at tasks requiring balance, strength, and coordination. They were also more inquisitive and apt to explore new environments, suggesting improved cognitive function. Physiological measures also pointed to better health: the knockout mice had stronger bones, better insulin sensitivity, and more robust immune cells. While male knockout mice did not have extended lifespans, they did have the same array of health benefits as females.

The effects of disabling S6K1 were similar to those of caloric restriction, though less pronounced. Female mice without S6K1 lived up to 20 percent longer than normal mice; the longevity increase with caloric restriction can reach 50 percent.”

Rice also notes that a study published in July showed that rapamycin—a drug used to prevent organ rejection in transplant patients and which interferes with the same pathway manipulated in the research just mentioned—also extends lifespan in mice. However, rapamycin also has potent immunosuppressant effects.

AutismRapamycin has its uses, however. It is one of three drugs being tested in humans to treat the results of genetic anomalies that cause Rett syndrome, fragile X, and tuberous sclerosis complex (TSC), all of which are often linked to autism. These are not true gene therapies—they do not target genes per se; rather, they are molecular therapies that fix problems caused by faulty genes.

Rapamycin has been shown to reduce seizures and abnormal brain enlargement as well as improve learning and memory in animals afflicted with TSC, which is caused by mutations in one of two genes that trigger development of benign tumors in the brain, eyes, heart, kidney, skin, and lungs. About 90 percent of TSC patients have epilepsy, and 50 percent have autism or other cognitive impairments. The drug is being tested to assess its effect on seizures, cognitive function, and other symptoms of autism.

Another drug targets a molecular receptor in Fragile X patients, who have a genetic mutation that results in over-stimulation of the receptor.

A third drug seeks to utilize IGF (insulin-like growth factor) and EGF (epidermal growth factor) pathways to fight Rett syndrome. Mice with the Rett mutation treated with a fragment of the IGF protein stimulated synapses, improved motor function, and extended life span. A clinical trial of IGF in girls ages 2 to 10 is planned or may already be underway.

BlindnessTwelve months after receiving an experimental gene therapy for Leber’s congenital amaurosis (LCA), three patients in their twenties who had been blind since birth were able to see brightly lit objects. A gene encoding a functional copy of a protein defective in LCA patients was injected into one eye of each patient. All three patients showed substantial improvements in their ability to detect light three months after treatment. After 12 months, one was able to read the LED dials in an automobile.

These results in restoring vision to patients who previously had no options for treatment have been called “spectacular” and “dramatic.” They may also “expedite development of gene therapy for more common retinal diseases, such as age-related macular degeneration.”

Color BlindnessA gene therapy has restored full color vision to adult male squirrel monkeys born without the ability to distinguish between red and green. More than two years ago, therapeutic genes containing DNA code enabling light-sensing cells to distinguish colors were implanted at the back of the eye of the monkeys, and the effect has not worn off.

“Further research is required, however, before this comes to human clinical trials, and therapy in the clinics,” a researcher said.

Cystic Fibrosis and Muscular DystrophyCystic fibrosis and muscular dystrophy are among several genetic disorders caused by mutations in genes that code for vital proteins. An oral small-molecule drug being developed by PTC Therapeutics modifies cellular machinery to restore production of normal proteins. The drug is now being tested in large, international clinical trials. Since it treats the underlying gene-expression problem, the drug could be effective in many diseases, including spinal muscular atrophy, hemophilia, and retinitis pigmentosa.

Results from early-stage clinical trials in cystic fibrosis and Duchenne muscular dystrophy patients were described as “incredibly encouraging” by a neurologist involved. About 50 percent of DMD patients taking the drug began making normal copies of a protein whose absence makes muscles fragile. Cystic fibrosis trial patients showed improved protein production and a 30 percent reduction in coughing.

Duchenne Muscular DystrophyA new technique called exon skipping has restored partial function in dogs with the canine form of Duchenne muscular dystrophy (DMD). “Traditional” gene therapy would seek to replace a mutated gene with a functional copy, whereas exon skipping relies on a variation of antisense, a technique in which short synthetic DNA or RNA molecules are designed to bind to a region of DNA or RNA and block its function.

Japanese and US scientists treated three canine DMD-afflicted beagles with weekly or biweekly intravenous injections of a cocktail of three different antisense molecules. After several weeks of treatment, the dogs showed noticeable improvements in muscle function tests and symptoms, and their cells produced dystrophin at an average of 26 percent of normal levels—similar to those found in human patients with BMD. It seems that while this approach would not cure the disease, it would reduce its severity.

Thus is the first time that researchers have successfully delivered an antisense therapy to alleviate DMD in a larger animal, but there remains much work to be done before it could be tested in humans

EpilepsyBy adding a normal Atp1a3 gene to epileptic mice in which the gene was defective, the mice’s progeny were born free of the disease. The human ATP1a3 gene is more than 99 percent the same as the mouse version. But as usual, a researcher cautioned “there’s a long way to go before this research could yield new antiepileptic therapies.”

The researchers found that an “intrabody” called Happ1 restored motor and cognitive function to the mice, and reduced neuron loss and toxic protein accumulation. In one type of mouse, Happ1 increased both body weight and life span.

Despite its single-gene origin, current treatments of the disease tend to address its symptoms rather than its cause.

The researchers are now working to increase Happ1’s efficacy and build a viral vector that can be turned off in case of unexpected side effects.

Other Neurological DisordersSeveral clinical trials are getting underway to test the precision-targeted delivery of genetic (and other) therapies directly to a specific part of the brain in Parkinson’s patients, using deep-brain stimulation (DBS) implants and advanced imaging. DBS reduces the symptoms of Parkinson’s but does not cure it.

In one small trial, a therapeutic gene called GAD, which stimulates production of a vital chemical missing in Parkinson’s patients, was delivered by catheter to the brain through a small hole drilled through the skull. Twenty-nine percent of the patients saw improvement in motor function, and a larger trial is either under way or may already have been completed.

Another trial using a similar technology inserts a gene that codes for a protein (GDNF, or glia-derived neurotropic factor) that enhances neuronal survival. Intra-operative neuroimaging helps the surgeon deliver the therapy with considerable precision. Researchers have already used this technique to deliver therapies to patients with brain cancer.

HIVIn February it was reported that a small (74 patient) phase I clinical trial of a gene therapy for HIV appeared to reduce the effect of the virus on the immune system. The molecule OZ1, which stops HIV from reproducing, was introduced into the patients’ blood stem cells.

After 100 weeks, the patients who received the gene therapy had higher levels of CD4+ cells (which HIV attacks) than the control group, which received a placebo, though it seems the effect was small and the therapy “is far from being perfected,” as one researcher put it.

In May, it was reported that a gene introduced via viral vector into the muscles of nine rhesus macaque monkeys produced antibody-like molecules that protected them from infection by SIV, the simian version of HIV.Four weeks after the therapy was administered (by direct injection into muscle), the researchers infected the monkeys with SIV. Six of the nine macaques showed no sign of the SIV infection, and the remaining three did not develop AIDS during the course of the study. In contrast, six control monkeys all became infected, and four of them died before the experiment finished.

The researchers are optimistic that their technique can be adapted to humans and hope to start clinical trials “soon.”

Hurler’s SyndromeA gene therapy for Hurler’s syndrome, the severe form of mucopolysaccharidoses (MPS), has shown considerable success in mice. Hurler’s and similar genetic disorders result from an inability to produce enzymes that help the body’s cells break down and recycle large molecules.

The therapy consists of two genes inserted via viral vector into hematopoietic stem cells in bone marrow. Human trials are a long way off.

The engineered mice “remained skinny despite the fact that they ate about the same and produced the same waste,” were as active as their normal counterparts, and showed no visible side effects. They also had lower fat levels in the liver, lower cholesterol levels, and did not convert the fat into sugar (which could lead to diabetes); rather, the excess fat was literally released into thin air as carbon dioxide.

Applying this method to humans is many steps away. But it’s a great advance.

Rheumatoid ArthritisMissing or mutated genes known as Foxp3 lead to autoimmune diseases including rheumatoid arthritis. A genetically engineered form of Foxp3 injected into animals has inhibited and even reversed the disease process. The researchers next plan to try to develop a Foxp3 drug for humans. Much work remains, however.

Sickle Cell DiseaseThere is hope of a cure for this devastating disease. An experimental therapy to implant (via viral vector) a gene that counteracts hemoglobin S was found, in animals and in human tissue samples, to prevent the production of the malformed red blood cells that characterize sickle cell disease. Clinical tests, in which the gene-carrying modified virus would be introduced into the patient’s bone marrow stem cells, could begin in 2010.

Almost There

The Common ColdIn February, researchers completed the DNA sequencing of all of the world’s 99 known strains of human rhinovirus—the virus responsible for the common cold. The sequences will reveal the virus’s vulnerabilities which could lead to development of cures, though not of a vaccine—the likelihood of mutations among so many strains is too great.

The sequences might also help find a cure or preventative for asthma, since rhinovirus in children appears able to reprogram the immune system to develop asthma by adolescence.

Mitochondrial DiseasesGenetic material needed to create a healthy baby has been successfully transferred from a defective monkey egg to a healthy one, resulting in healthy births. The technique involved taking genetic material from healthy mitochondria in the donor’s egg to replace faulty mitochondria in the recipient’s egg. Babies born with faulty mitochondria (which have their own DNA, separate from the cell’s human nuclear DNA) can develop many different diseases including anemia, dementia, hypertension, and neurological disorders. Nearly 250 mitochondrial DNA mutations have been shown to cause or contribute to human diseases.

The technique is raising perplexed eyebrows among ethicists, since the changes are passed on down the generations yet they do not involve changing nuclear DNA—the strictly human side of the germline, which some consider an ethical no-go area.

The researchers think they could be ready for human clinical trials within two to three years.

ParalysisStartup firm LucCell will commercialize a molecular “light switch” able to control individual neurons in animals genetically modified (via a viral vector) to respond to the switch. LucCell will initially develop switches to activate the diaphragm or the external sphincter muscle in paralyzed people, thereby restoring breathing or bladder control respectively. The light source will be a miniature laser or LED implant attached to an optical fiber that runs to the muscle to be controlled.

The technology is already in widespread use in basic science, including to study psychiatric and neurological disorders as well as normal brain functions, and it has already succeeded in restoring breathing function in paralyzed rodents, even lasting for a day after the switch was turned off. Another startup firm will use the technology to develop therapies for blindness.

There remain several years of development and testing before such a therapy reaches the market.

Fanconi AnemiaSkin cells from six Fanconi anemia patients were given a functional copy of one of the faulty genes responsible for the rare condition, which causes skeletal problems and bone-marrow failure, and elevates the risk of cancer. The cells were then transformed into induced pluripotent stem cells (iPSCs) capable of growing into any tissue type—including the healthy blood cells needed to correct the patients’ inherited anemia.

The theory is that the genetically corrected iPSCs would generate a supply of healthy blood cells if transplanted back into the patients, though that step was not taken in the reported study because there remain many unknowns to be researched and because the iPSCs were not specifically designed for transplantation. Rather, this was a proof-of-concept study to confirm the viability of producing genetically corrected iPSCs. Its success shows that iPSC therapy has great promise to cure and even reverse the effects of disease.

A press release from the Salk Institute for Biological Studies said the study “has catapulted the field of regenerative medicine significantly forward, proving in principle that a human genetic disease can be cured using a combination of gene therapy and induced pluripotent stem (iPS) cell technology.” A Salk researcher added: “We haven’t cured a human being, but we have cured a cell. In theory we could transplant it into a human and cure the disease.”

InfertilityA common factor in infertility is poor quality egg or sperm. Early this year, scientists used iPSCs to produce healthy egg- and sperm-cell precursors. This was the first time that iPSCs have been turned into germline cells—previously, only somatic cells had been produced.

However, the new method is “many, many, many years away from generating a cell type that would be capable of fertilization and, therefore, making a healthy child,” the researchers caution. Furthermore, using embryonic stem cells rather than iPSCs resulted in “substantially healthier” egg and sperm precursors with fewer chromosomal abnormalities.

More Biomarkers

Esophageal CancerOne recently discovered biomarker indicates a possible precancerous condition in the esophagus called Barrett’s esophagus (BE) which increases the risk of developing esophageal adenocarcinoma (EAC), the most common form of esophageal cancer. Standard pathology has difficulty identifying the presence of BE.

Cause è Potential Gene (and other) Therapy for PAPA familial genetic mutation has been found to cause an inherited form of the lung disease pulmonary alveolar proteinosis (PAP). Patients with PAP suffer labored breathing and respiratory failure, and are susceptible to secondary infections. The findings provide a basis for developing a better therapy than whole-lung wash, where a tube is inserted into the airway under anesthesia to flush out the lungs.

The discoverers of the genetic link think that an inhaled aerosol drug, or a bone marrow transplant, or a gene therapy might be effective, and are pursuing all three approaches.

Odds & Ends

Important New Research Accelerant from ProteomicsEarlier this year saw the creation of the first genetically modified mammals developed using zinc finger nuclease (ZFN) technology. ZFNs are engineered proteins that act somewhat like knives to cut out (or “knock out” as the scientists like to say) pieces of DNA. The modified rats were given permanent, heritable gene mutations, paving the way for the rapid development of novel genetically modified animal models of human disease. ZFN technology speeds things up by bypassing the current need to conduct cumbersome experiments involving nuclear transfer (cloning) or embryonic stem cells.

About 90 percent of the rat’s 25,000-30,000 estimated genes are analogous to those in humans and mice, and their larger size makes them a superior model for drug-evaluation studies. The new technique will increase the rat’s usefulness in research pertaining to physiology, endocrinology, neurology, metabolism, parasitology, growth and development, and cancer.

A biotechnology company is already using the technology to develop a rat-based human antibody platform, by knocking out the gene encoding rat immunoglobulin M (IgM), an important gene for rat antibody production.

Personalized MABsSeveral monoclonal antibodies or “MABs” (drugs engineered to precisely target specific diseased cell molecules and destroy the diseased cell) are approved for treating cancers and autoimmune diseases, and nearly 200 are in clinical trials. But some patients don’t respond well or at all, and it appears that their genotype is the problem. A startup called PIKAMAB is developing MABs customized for the genotype.

PIKAMAB is developing a “diapeutic” (or “theranostic”) test that separates patients into one of nine genotypes ranging from those likely to respond well to an existing drug to those who are likely to respond poorly. Antibodies customized for each group would then be delivered accordingly.

There is some skepticism about this approach—there are alternatives—but at least it is a step in the direction of personalized medicine.

Personalized Medicine In Practice at Mass GenEarlier this year, Massachusetts General Hospital began to read the genetic fingerprints of nearly all new cancer patients’ tumors—that’s 5-6,000 patients a year, with a view to offering personalized therapies. A robotic system will look for 110 abnormalities, carried on 13 major cancer genes, that can predict whether drugs already on the shelf or in development might work.

The state’s three major health plans apparently will pay for such testing only when it has proven medical benefits. Otherwise, the hospital must absorb the cost or seek payment from patients. Eventually, however, such screening will be commonplace.

Genetically Modified Health FoodLast year, scientists created purple tomatoes rich in an antioxidant pigment called anthocyanin, found in high levels in blackberry, cranberry, chokeberry, and other berries and thought to have anti-cancer properties. Experimental mice that ate the tomatoes lived longer.

Anthocyanins have been shown to help significantly slow the growth of colon cancer cells, and may protect against cardiovascular disease and age-related degenerative diseases, have anti-inflammatory properties, help boost eyesight, and help prevent obesity and diabetes.

With such concentrated benefit, the fruit would be help the many people who do not eat the recommended daily amount and types of vegetables.

Monitoring Therapy BreakthroughBack in 2008, a way was found to genetically re-engineer therapeutic T cells so that they absorb an imaging agent and hold it for life—and beyond, if they divide. The method should enable physicians and researchers to track precisely what every individual T cell does, and where, and thus know precisely how well a T cell therapy is working.

The method was tested in a patient with an aggressive brain tumor (glioblastoma) enrolled in a clinical trial of cell-based therapy. “The cells were actually good at finding the tumor,” the lead researcher said in a press release, who pointed out that the same technique could be used to follow other immune cells or eventually stem cells throughout the body.

DIY GenomicsHobbyists (and Lord knows who else) continue to engineer synthetic life forms, using lab equipment bought on Craigslist or eBay or made at home, and informed by Googled knowledge. One hobbyist is trying to develop, pro bono publico, genetically altered yogurt bacteria that will glow green if melamine, the chemical that turned Chinese-made baby formula and pet food deadly, is present.

But even the best-intentioned “biohackers” could cause environmental or medical catastrophe. Many, according to AP writer Marcus Wohlsen, have no advanced degree and no experience in the biotechnology field.

Such instances point to one of the key issues arising from the accelerating development of genomic science and medicine to which we alluded at the beginning of this issue. We will report further on this and other key issues.